Identification of a genetic mutation in a family with fructose-1,6- bisphosphatase deficiency

Fructose-1,6-bisphosphatase deficiency is an inheritable disorder of gluconeogenesis. Sequence analysis of the cDNA of the fructose-1,6-bisphosphatase mRNA isolated from monocytes from a girl with this disease and her consanguineous parents revealed that the patient and her parents were a homozygote and heterozygotes for an insertion of one G residue at G957GGGG961, respectively. This mutation resulted in translation of a truncated enzyme protein, and the mutant protein showed no fructose-1,6- bisphosphatase activity in an overexpression experiment in Escherichia coli. However, this mutation is located in a region of the amino acid sequence which is not well conserved among mammals. A mutagenized clone was prepared from the normal clone. The extents of substitutions and deletions of the amino acid sequence were predicted to be less in the mutagenized protein than in the mutant protein. This mutagenized clone also expressed no fructose-1,6-bisphosphatase activity, although both of two normal clones from control monocytes and a control liver sample expressed an apparently normal level of fructose-1,6-bisphosphatase activity. Thus, this mutation is concluded to be responsible for fructose-1,6-bisphosphatase deficiency in this patient.     

Function, structure and evolution of fructose-1,6-bisphosphatase

The hydrolysis of fructose-1,6-bisphosphate to fructose-6-phosphate is a key reaction of carbohydrate metabolism. The enzyme that catalyzes this reaction, fructose-1,6-bisphosphatase, appears to be present in all forms of living organisms. Regulation of the enzyme activity, however, occurs by a variety of distinct mechanisms. These include AMP inhibition (most sources), cyclic AMP-dependent phosphorylation (yeast), and light-dependent activation (chloroplast). In this short review, we have analyzed the function of several fructose-1,6-bisphosphatases and we have made a comparison of partial amino acid sequences obtained from the enzymes of the yeast Saccharomyces cerevisiae, Escherichia coli, and spinach chloroplasts with the known entire amino acid sequence of a mammalian gluconeogenic fructose-1,6-bisphosphatase. These results demonstrate a very high degree of sequence conservation, suggesting a common evolutionary origin for all fructose-1,6-bisphosphatases.     

Regulation of fructose-1,6-bisphosphatase activity in primary cultured astrocytes

In the gluconeogenic pathway, fructose-1,6-bisphosphatase (EC 3. 1. 3. 11) is the last key-enzyme before the synthesis of glucose-6-phosphate. The extreme diversity of cells present in the whole brain does not facilitate in vivo study of this enzyme and makes it difficult to understand the regulatory mechanisms of the related carbohydrate metabolism. It is for instance difficult to grasp the actual effect of ions like potassium, magnesium and manganese on the metabolic process just as it is difficult to grasp the effect of different pH values and the influence of glycogenic compounds such as methionine sulfoximine. The present investigation attempts to study the expression and regulation of fructose-1,6-bisphosphatase in cultured astrocytes. Cerebral cortex of new-born rats was dissociated into single cells that were then plated. The cultured cells were flat and roughly polygonal and were positively immunostained by anti-glial fibrillary acidic protein antibodies. Cultured astrocytes are able to display the activity of fructose-1,6-bisphosphatase. This activity was much higher than that in brain tissue in vivo. Fructose-1,6-bisphosphatase in cultured astrocytes did not require magnesium ions for its activity. The initial velocity observed when the activity was measured in standard conditions was largely increased when the enzyme was incubated with Mn2+. This increase was however followed by a decrease in absorbance resulting in the induction, by the manganese ions, of a singular kinetics in the enzyme activity. Potassium ions also stimulated fructose-1,6-bisphosphatase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hypoglycaemic effect of AICAriboside in mice

We have previously demonstrated that in isolated hepatocytes from fasted rats, AICAriboside (5-amino 4-imidazolecarboxamide riboside), after its conversion into AICAribotide (AICAR or ZMP), exerts a dose-dependent inhibition on fructose-1,6-bisphosphatase and hence on gluconeogenesis. To assess the effect of AICAriboside in vivo, we measured plasma glucose and liver metabolites after intraperitoneal administration of AICAriboside in mice. In fasted animals, in which gluconeogenesis is activated, AICAriboside (250 mg/kg body weight) induced a 50% decrease of plasma glucose within 15 min, which lasted about 3 h. In fed mice, glucose decreased by 8% at 30 min, and normalized at 1 h. Under both conditions, ZMP accumulated to approximately 2 mumol/g of liver at 1 h. It decreased progressively thereafter, although much more slowly in the fasted state. Inhibition of fructose-1,6-bisphosphatase was evidenced by time-wise linear accumulations of fructose-1,6-bisphosphate, from 0.006 to 3.9 mumol/g of liver at 3 h in fasted mice, and from 0.010 to 0.114 mumol/g of liver at 1 h in fed animals. AICAriboside did not significantly influence plasma insulin or glucose utilization by muscle. We conclude that in vivo as in isolated hepatocytes, AICAriboside, owing to its conversion into ZMP, inhibits fructose-1,6-bisphosphatase and consequently gluconeogenesis.     

A study of subunit interface residues of fructose-1,6-bisphosphatase by site-directed mutagenesis: effects on AMP and Mg2+ affinities

The structural transformation of fructose-1,6-bisphosphatase upon binding of the allosteric regulator AMP dramatically changes the interactions across the C1-C4 (C2-C3) subunit interface of the enzyme. Asn9, Met18, and Ser87 residues were modified by site-directed mutagenesis to probe the function of the interface residues in porcine liver fructose-1,6-bisphosphatase. The wild-type and mutant forms of the enzyme were purified to homogeneity and characterized by initial rate kinetics and circular dichroism (CD) spectrometry. No discernible alterations in structure were observed among the wild-type and Asn9Asp, Met18Ile, Met18Arg, and Ser87Ala mutant forms of the enzyme as measured by CD spectrometry. Kinetic analyses revealed 1.6- and 1.8-fold increases in kcat with Met18Arg and Asn9Asp, respectively. The K(m) for fructose 1,6-bisphosphate increased about 2-approximately 4-fold relative to that of the wild-type enzyme in the four mutants. A 50-fold lower Ka value for Mg2+ compared with that of the wild-type enzyme was obtained for Met18Ile with no alteration of the Ki for AMP. However, the replacement of Met18 with Arg caused a dramatic decrease in AMP affinity (20 000-fold) without a change in Mg2+ affinity. Increases of 6- and 2-fold in the Ki values for AMP were found with Asn9Asp and Ser87Ala, respectively. There was no difference in the cooperativity for AMP inhibition between the wild-type and the mutant forms of fructose-1,6-bisphosphatase. This study demonstrates that the mutation of residues in the C1-C4 (C2-C3) interface of fructose-1,6-bisphosphatase can significantly affect the affinity for Mg2+, which is presumably bound 30 A away. Moreover the mutations alternatively reduce AMP and Mg2+ affinities, and this finding may be associated with the destabilization of the corresponding allosteric states of the enzyme. The kinetics and structural modeling studies of the interface residues provide new insights into the conformational equilibrium of fructose-1,6-bisphosphatase.     

A comparison of gene organization in the zwf region of the genomes of the cyanobacteria Synechococcus sp. PCC 7942 and Anabaena sp. PCC 7120

The region of the genome encoding the glucose-6-phosphate dehydrogenase gene zwf was analysed in a unicellular cyanobacterium, Synechococcus sp. PCC 7942, and a filamentous, heterocystous cyanobacterium, Anabaena sp. PCC 7120. Comparison of cyanobacterial zwf sequences revealed the presence of two absolutely conserved cysteine residues which may be implicated in the light/dark control of enzyme activity. The presence in both strains of a gene fbp, encoding fructose-1,6-bisphosphatase, upstream from zwf strongly suggests that the oxidative pentose phosphate pathway in these organisms may function to completely oxidize glucose 6-phosphate to CO2. The amino acid sequence of fructose-1,6-bisphosphatase does not support the idea of its light activation by a thiol/disulfide exchange mechanism. In the case of Anabaena sp. PCC 7120, the tal gene, encoding transaldolase, lies between zwf and fbp.     

Urinary sugar phosphates and related organic acids in fructose-1,6-diphosphatase deficiency

Two sisters with fructose-1,6-diphosphatase deficiency are reported. They presented with ketonuria, elevated plasma transaminase activity and severe metabolic acidosis during hypoglycaemic crises, which resembled Reye syndrome. Intravenous fructose tolerance tests provoked severe hypoglycaemia and metabolic acidosis. Fructose-1,6-diphosphatase activities in both peripheral leukocytes and cultured lymphocytes were below the limit of detection. Urinary organic acid analysis during crises revealed markedly increased excretion of lactate, ketone bodies, glycerol and glycerol-3-phosphate. We newly identified other glycolytic intermediates, glyceraldehyde, 3-phosphoglycerate and fructose-1,6-diphosphate, in the urine during hypoglycaemic attacks or after fructose tolerance tests. Identification of such compounds may be useful in the early diagnosis of this disease.     

Effects of different hybrids, strains and age of laying hens on the cholesterol content of the table egg

The intrinsic fluorescence of homogeneous castor oil seed cytosolic fructose-1,6-bisphosphatase (FBPasec) was used as an indicator of conformational changes due to ligand binding. Binding of the substrate and the inhibitor fructose-2,6-bisphosphate (F-2,6-P2) was quantitatively compared to their respective kinetic effects on enzymatic activity. There are two distinct types of substrate interaction with FBPasec, corresponding to catalytic and inhibitory binding, respectively. Inhibitory substrate binding shares several characteristics with F-2,6-P2 binding which indicates that both ligands bind at the same site. However, F-2,6-P2 does not prevent fluorescence transitions attributed to catalytic substrate binding. The marked synergistic inhibition of FBPasec by AMP and F-2,6-P2 appears to arise via AMP's promotion of F-2,6-P2 binding. Based on the X-ray crystal structure of porcine kidney FBPase our modelling studies suggest the existence of a distinct F-1,6-P2/F-2,6-P2 inhibitory binding site which partially overlaps with the enzyme's catalytic site. We propose that a pronounced allosteric transition mediated by AMP binding increases access of F-1,6-P2 and F-2,6-P2 to this common inhibitory binding site.     

Role of electrostatic interactions on the affinity of thioredoxin for target proteins. Recognition of chloroplast fructose-1, 6-bisphosphatase by mutant Escherichia coli thioredoxins

Chloroplast thioredoxin-f functions efficiently in the light-dependent activation of chloroplast fructose-1, 6-bisphosphatase by reducing a specific disulfide bond located at the negatively charged domain of the enzyme. Around the nucleophile cysteine of the active site (-W-C-G-P-C-), chloroplast thioredoxin-f shows lower density of negative charges than the inefficient modulator Escherichia coli thioredoxin. To examine the contribution of long range electrostatic interactions to the thiol/disulfide exchange between protein-disulfide oxidoreductases and target proteins, we constructed three variants of E. coli thioredoxin in which an acidic (Glu-30) and a neutral residue (Leu-94) were replaced by lysines. After purification to homogeneity, the reduction of the unique disulfide bond by NADPH via NADP-thioredoxin reductase proceeded at similar rates for all variants. However, the conversion of cysteine residues back to cystine depended on the target protein. Insulin and difluoresceinthiocarbamyl-insulin oxidized the sulfhydryl groups of E30K and E30K/L94K mutants more effectively than those of wild type and L94K counterparts. Moreover, the affinity of E30K, L94K, and E30K/L94K E. coli thioredoxin for chloroplast fructose-1,6-bisphosphatase (A0.5 = 9, 7, and 3 microM, respectively) increased with the number of positive charges, and was higher than wild type thioredoxin (A0.5 = 33 microM), though still lower than that of thioredoxin-f (A0.5 = 0.9 microM). We also demonstrated that shielding of electrostatic interactions with high salt concentrations not only brings the A0.5 for all bacterial variants to a limiting value of approximately 9 microM but also increases the A0.5 of chloroplast thioredoxin-f. While negatively charged chloroplast fructose-1,6-bisphosphatase (pI = 4.9) readily interacted with mutant thioredoxins, the reduction rate of rapeseed napin (pI = 11.2) diminished with the number of novel lysine residues. These findings suggest that the electrostatic interactions between thioredoxin and (some of) its target proteins controls the formation of the binary noncovalent complex needed for the subsequent thiol/disulfide exchange.     

Solitary fibrous tumor arising from hyperplastic thymus

In a population based study, the prescribed insulin dose of 348 prepubertal children with insulin-dependent diabetes mellitus (IDDM) was analysed 2 years after the diagnosis of diabetes. Girls had an insulin dose 13.6% higher than that in boys. When children younger than 5 years of age at diagnosis were analysed separately, the difference in insulin dose between boys and girls remained. The increased insulin dose in girls was not explained by possible differences in endogenous insulin secretion, body mass index, metabolic control or the number of daily insulin injections. Our observations indicate that prepubertal girls with IDDM have a poorer insulin sensitivity than boys.     

The diaphragm and respiratory muscles

A rapid procedure for the purification of the redox-regulated chloroplast fructose-1,6-bisphosphatase [EC 3.1.3.11] from spinach leaf extract to homogeneity is described. No thiol-reducing agents were present during the purification and the enzyme is > 99% in the oxidized form. A rapid procedure to reduce and activate the Fru-1,6-P2ase by dithiothreitol in the absence of thioredoxin is described. Reduction activates the enzyme up to several hundred-fold when assayed at pH 8.0 with 2 mM Mg2+. The activity of the purified oxidized enzyme is unusually sensitive to changes in Mg2+ and H+ concentration. Tenfold changes in Mg2+ or H+ concentration lead to > 100-fold increases in activity. The recoveries of fructose-1,6-bisphosphatase activity as determined by the activity of the oxidized enzyme at pH 8.0/20 mM Mg2+; pH 9.0/2 mM Mg2+; pH 8/2 mM Mg2+ plus 0.1 mM Hg(II) or of the reduced enzyme at pH 8.0/2 mM Mg2+ are similar (approximately 40%) indicating that the major proportion of these activities in a leaf extract is catalyzed by the same enzyme. Moreover, antibodies raised against the purified enzyme inhibit all of the above activities in crude leaf extracts. The kinetic properties of the purified enzyme suggest that the oxidized Mg(2+)-dependent enzyme can play no significant role in photosynthetic carbon assimilation. A survey of some kinetic properties of Fru-1,6-P2ase activity in extracts of various photosynthetic organisms reveals that all 11 species examined possess a redox- and pH/Mg(2+)-stimulated Fru-1,6-P2ase, whereas Fru-1,6-P2ase in extracts of Taxus baccata (a gymnosperm), Chlorella vulgaris (a green alga), and the cyanobacterium Nostoc muscorum were not activated by Hg(II). The heat stability that proved useful in the purification of the spinach enzyme was conserved in both angiosperms and gymnosperms. The oxidized enzyme (which normally has no thiol groups accessible to 5,5'-dithio-bis[2-nitrobenzoic acid]) but not the reduced enzyme can be stimulated many hundred-fold by addition of extraordinarily low concentrations of Hg(II) to a complete assay mixture. With the aid of EDTA as a Hg(II) buffer, half-maximal stimulation was achieved at 2 x 10(-16) M free Hg(II). Methylmercury also stimulates the enzyme many hundred-fold at very low concentrations. The concentration for half-maximal stimulation by methylmercury was determined with a cyanide buffer to be approximately 10(-16) M. This, together with the high affinity of the enzyme for Hg(II), suggests that Hg(II) stimulates the enzyme by binding to an enzyme thiol group that be comes exposed in the catalytically active enzyme, thereby stabilizing the oxidized enzyme in an active conformation. By contrast, in the absence of Fru-1,6-P2 and either Mg2+ or Ca2+, Hg(II) (even at 2 x 10(-16) M) rapidly inactivates the oxidized Fru-1,6-P2ase. This inactivation is similar to the inactivation of Fru-1,6-P2ase that occurred at high pH (> 9) and which is also prevented by Fru-1,6-P2 and either Mg2+ or Ca2+. Although the Hg(II)- and high pH-inactivated oxidized enzyme has no activity, both forms of the enzyme can be activated by reduction. The usefulness of buffers to maintain low, defined Hg(II) and organic mercurial concentrations is discussed.     

Human gallbladder mucosal function: effects on intraluminal fluid and lipid composition in health and disease

OBJECTIVES: Fructose-1,6-diphosphate is a glycolytic intermediate that has been shown experimentally to cross the cell membrane and lead to increased glycolytic flux. Because glycolysis is an important energy source for myocardium during early reperfusion, we sought to determine the effects of fructose-1,6-diphosphate on recovery of postischemic contractile function. METHODS: Langendorff-perfused rabbit hearts were infused with fructose-1,6-diphosphate (5 and 10 mmol/L, n = 5 per group) in a nonischemic model. In a second group of hearts subjected to 35 minutes of ischemia at 37 degrees C followed by reperfusion (n = 6 per group), a 5 mmol/L concentration of fructose-1,6-diphosphate was infused during the first 30 minutes of reperfusion. We measured contractile function, glucose uptake, lactate production, and adenosine triphosphate and phosphocreatine levels by phosphorus 31-nuclear magnetic resonance spectroscopy. RESULTS: In the nonischemic hearts, fructose-1,6-diphosphate resulted in a dose-dependent increase in glucose uptake, adenosine triphosphate, phosphocreatine, and inorganic phosphate levels. During the infusion of fructose-1,6-diphosphate, developed pressure and extracellular calcium levels decreased. Developed pressure was restored to near control values by normalizing extracellular calcium. In the ischemia/reperfusion model, after 60 minutes of reperfusion the hearts that received fructose-1,6-diphosphate during the first 30 minutes of reperfusion had higher developed pressures (83 +/- 2 vs 70 +/- 4 mm Hg, p < 0.05), lower diastolic pressures (7 +/- 1 vs 12 +/- 2 mm Hg, p < 0.05), and higher phosphocreatine levels than control untreated hearts. Glucose uptake was also greater after ischemia in the hearts treated with fructose-1,6-diphosphate. CONCLUSIONS: We conclude that fructose-1,6-diphosphate, when given during early reperfusion, significantly improves recovery of both diastolic and systolic function in association with increased glucose uptake and higher phosphocreatine levels during reperfusion.     

An ADP-ribosylation-factor(ARF)-like protein involved in regulated secretion

In higher plants, light enhances the activity of chloroplast fructose-1,6-bisphosphatase via a cascade of thiol/disulfide exchanges. We have examined the structural and functional role of seven conserved cysteine residues in the rapeseed (Brassica napus) enzyme by site-directed mutagenesis. After lysis of Escherichia coli cells, C53S and C191S variants partitioned mainly in the insoluble fraction whereas C96S, C157S, C174S, C179S, and C307S mutants were soluble. Homogeneous preparations of the latter hydrolyzed fructose 1,6-bisphosphate at similar rates in the presence of 10 mM Mg2+ but only C157S, C174S and C179S mutants were both efficient catalysts at 1 mM Mg2+ and nearly insensitive to dithiothreitol. These results demonstrate the contribution of Cys53 and Cys191 to the stability of the enzyme and the participation of Cys157, Cys174 and Cys179 in the reductive process responsive of the light-dependent regulation. Given that mutations at Cys96 and Cys307 neither destabilize the enzyme nor affect the reductive modulation, their function remains unknown.     

Crystal structure of a trapped phosphoenzyme during a catalytic reaction

The crystal structure of the fructose-2,6-bisphosphatase domain trapped during the reaction reveal a phosphorylated His 258, and a water molecule immobilized by the product, fructose-6-phosphate. The geometry suggests that the dephosphorylation step requires prior removal of the product for an 'associative in-line' phosphoryl transfer to the catalytic water.     

HLA-DRB1 and tumor necrosis factor gene polymorphisms in Japanese patients with multiple sclerosis

A significant difference between cytosolic and chloroplastic fructose-1,6-bisphosphatase (FbPase) is an extra peptide in the middle of chloroplast FbPase which contains three additional cysteine residues. Sit-directed mutagenesis experiments have shown that at least two of these cysteine residues are involved in forming the regulatory disulfide bridge [Jacquot, J.-P. et al., FEBS Lett. 401 (1997) 143-147] which is the presupposition for the thioredoxin-dependent control of chloroplast FbPase activity. Here we report that each subunit of the FbPase contains an additional structural disulfide bridge which has been observed by combined application of thioredoxins and sulfitolysis. Observation of the structural disulfide bridges by sulfitolysis was only possible when the FbPase was already specifically reduced by the homologous thioredoxin species TRm. and TRf from spinach chloroplasts. Interestingly, the accessibility of the structural disulfide bridge for sulfite ions depends on the thioredoxin species engaged in the thioredoxin/FbPase complex.     

Peak expiratory flow rate in Nigerian school children

Peak expiratory flow rate (PEFR) was measured using the Wright's peak flow meter in 263 school boys and 275 school girls living in Lagos, Nigeria. Their ages ranged from 6.0 years to 19.0 years (mean 11.9 +/- 3.8 yrs. for boys and 11.8 +/- 3.9 yrs. for girls). Mean PEFR was 359.2 +/- 102.0 L/min (range 160.0-610.0 L/min) in boys and 327.7 +/- 81.3 L/min (range 160.0-500.0 L/min) in girls. Apart from ages 17, 18 and 19, peak expiratory flow rates were similar in both boys and girls. In both sexes, PEFR correlated positively and significantly with age, height, weight and body surface area. Also in both sexes and in all age groups studied, PEFR was significantly higher than predicted values obtained from previous Nigerian and caucasian prediction equations. When compared with values obtained from a second caucasian prediction equation, observed values were significantly higher in the 6-10 years and 16-19 years age groups in boys and 11-15 years and 16-19 years age groups in girls. New prediction equations for calculating PEFR in Nigerian boys and girls are presented. Observed PEFR may be due to enhanced stature in Nigerian children resulting from improved environment and genetic factors.     

Age at onset of puberty following high-dose central nervous system radiation therapy

OBJECTIVE: To determine if a relationship exists between age at irradiation, sex of the patient, and age at onset of puberty and pubarche in children treated with high-dose radiation to the central nervous system. DESIGN: Case series. SETTING: Tertiary care institutional practices and clinics. PATIENTS: Thirty-six children treated with high-dose irradiation (hypothalamic pituitary dose, 30-72 Gy) by conventional (n = 29) or hyperfractionated (n = 7) schedules. Girls were treated before age 8 years and boys before age 9 years. Twenty-six of the 36 children also received chemotherapy. All tumors were distant from the hypothalamic-pituitary region. MAIN OUTCOME MEASURE: Age at onset of puberty and pubarche. RESULTS: In girls, the median age at onset of puberty was 9.3 years vs 10.9 years for controls (P < .01); pubarche occurred at 9.4 years vs 11.2 years for controls (P < .01). In boys, the median age at onset of puberty--genital II--was 11.0 years vs 11.5 years for controls (P = .30); pubarche occurred at a median age of 10.5 years vs 12 years for controls (P = .25). A censored-data normal linear regression model was used to account for children (n = 6) who had not reached puberty. Age at diagnosis (P < .01) and sex (P = .01) were significant predictors of age at onset of puberty. Body mass index SD score (z score) was inversely related to age at onset of puberty (r = -0.77) and was greater at onset of puberty in girls than in boys. CONCLUSION: In children who have received high-dose cranial radiation therapy, a significant positive correlation exists between age at diagnosis and age at onset of puberty in boys and girls.